DE60034834T2 - Transformed brassica plant - Google Patents

Abstract

A transformed plant is described. The transformed plant comprises an exogenous transparent seed coat gene obtained from an AA genome. Also disclosed is a method for increasing the levels of seed oil and protein and reducing the levels of fibre in a seed by transferring the transparent seed coat gene of an AA genome of a first Brassica plant into a CC genome of a second Brassica plant.

Description

The The present invention relates to an organism. In particular, it concerns the present invention, a transformed plant and tissue or Cells thereof and transformed cells or tissues.

The Species Brassica napus (AACC, 2n = 38) is an amphidiploid species, in antiquity from the diploid species B. campestris (AA, 2n = 20) and B. oleracea (CC, 2n = 18) by interspecific hybridization emerged. All of course occurring strains of B. napus (which also carries the oilseed Include rapeseed) are of the type with black seeds.

In The species Brassica is the yellow seed of the brown / black seed due to its higher oil and protein content, its lower fiber content and its aesthetic appearance in seed-containing Superior to food. For example, the yellow or partially yellow seed of B. campestris has a 2-5% higher oil content, 1% higher Protein content and 4-7% lower fiber content (Stringam et al 1974, Daun and DeClercq 1988, Hu 1988; Simbaya et al. 1995), the yellow seed of B. juncea brings a 2.8% higher oil content, a 3.5% higher protein content and a 7.3% lower fiber content (Woods 1980, Simbaya et al. 1995), the yellow seed of B. carinata yields a 3.8% higher Protein content and a 5.5% lower fiber content (Simbaya et al. 1995) and the seeds of B. napus with brown or yellow-brown Seeds developed by crossing contain a 2-4% higher oil and protein content and a 3-7% lower fiber content (Shirzadegan and Roebbelen 1985; Hu 1988; Simbaya et al. 1995).

There most seed oil and protein is localized in the embryo, the seed coat contains very small Quantities of oil and protein. About that In addition, the proportion of seed coat relative to the total seed yellow seed is 15% lower than brown-seeded rapeseed (B. campestris) (Stringam et al., 1974). Accordingly, the higher oil and protein content in yellow seed predominantly a consequence of the larger portion of embryo in the Compared to the overall name. The lower fiber content in of yellow or weakly colored Seed-derived food is largely a consequence of a lower one Proportion of shell, compared to the total seed (Stringam et al. 1974; Anjou et al. 1977).

The Pigment of the seed coat of Brassica consists mainly of Polyphenols, which are polymers of leucocyanidines (Leung et al. 1979; Hu 1988). The polyphenyl is found in the palisade and in the compressed Parenchyma layers of the seed coat deposited (Vaugan 1970, Stringam et al. 1974). The palisade layer is the salient cell layer the seed coat, and is believed to have a high fiber content and has a lower oil content and protein content. The palisade cell layer is in the yellow seed of beet rape reduced by 1/2 to 2/3 (Stringam et al., 1974). Accordingly the yellow seed will have lower levels of polyphenol and lignin (Anjou et al 1977, Theander et al 1977, Slominski et al. 1994). As a result, the digestibility of the seed coat or of foods from yellow seed significantly higher than that of black seeds (Bell and Shires 1982, Slominski et al., 1994).

in view of the aforementioned benefits associated with yellow seeds, would it be desirable, to produce transformed cells that are able to seed with to provide yellow color and / or plants with yellow seeds.

One transformed CC genome, which is an exogenous gene for a transparent Seed coat obtained from an AA genome is included taught here. The gene for a transparent seed coat provides a seed with yellow color since the shell is transparent, thereby allowing the yellow interior of the seed can be seen. The visible yellow seed is produced by a substantially transparent seed coat, which thereby the natural one Embryo opposite the bare one Eye exposed.

Of the Term "exogenous Gene for a transparent seed coat "means as used herein, a gene for a transparent seed coat, which a different origin than the other genes that on a particular genome.

The CC genome is found in some Brassica. Accordingly, the CC genome available from Brassica. Typically, the CC genome Brassica is obtained. In other words the CC genome is a Brassica CC genome.

The AA genome is found in some Brassica. Accordingly, the AA genome available from Brassica. Typically, the AA genome is obtained from Brassica. In other words the AA genome is a Brassica AA genome. Preferably, the AA genome from any one among Brassica campestris, Brassica napus and Received Brassica juncea.

Preferably the AA genome is obtained from Brassica campestris.

Different expressed herein is taught a transformed Brassica CC genome, wherein the transformed Brassica CC genome is a gene for a transparent seed coat comprises which was obtained from a Brassica AA genome.

Preferably For example, the CC genome is a transformed Brassica napus genome.

Of the Visible yellow seeds are produced by a substantially transparent Seedshell, thereby the natural embryo to the naked eye exposed. This is in direct opposition to the seeds of Brassica plants with black seeds and those with yellow-brown ones Seeds that show darker colors as a result of the darker coloration and the dark pigmentation in the seed coat.

According to the present Invention includes the Term "gene for a transparent Seed coat "- which can be exchanged with the term "gene for a transparent seed coat color" or the term "gene for a yellow Seed coat "or the Term "gene for a transparent yellow seed coat "- one or several nucleotide sequences, but preferably at least two Nucleotide sequences that are capable of a transparent seed coat to rent. If there are at least two nucleotide sequences, then can these at the same chromosomal site or at different chromosomal sites To be localized.

Therefore the term "gene for a transparent Seed coat "the alternatively be given for a gene or a number of genes that are capable of producing a seed coat essentially transparent and thereby the visualization the essentially natural to allow yellow color of the internal embryo component of the semen.

A The seed coat is substantially transparent when seed coat pigmentation essentially not compared to the seed coat of the wild-type is available.

Of the Term 'wild type' means as well as he is used here, a form of a genallel, which is considered the "standard" or the most common is found in nature to be found type.

The present invention discloses a transformed Brassica plant, a plant cell or plant tissue with a Brassica CC genome, which is an exogenous gene for a transparent seed coat obtained from an AA genome was, includes, wherein the transformed from this plant, plant cell or the transformed plant tissue obtained a consistent and have stable yellow color.

Preferably is the transformed plant, plant cell or the transformed one Plant tissue which is an exogenous gene for the transparent seed coat includes, received from the AA genome of any one among Brassica campestris, Brassica napus and Brassica juncea.

Preferably is the transformed plant, plant cell or the transformed one Plant tissue which is an exogenous gene for the transparent seed coat comprises obtained from the AA genome of Brassica campestris.

Preferably is the transformed plant, plant cell or the transformed Plant tissue of a transformed Brassica napus plant, plant cell or plant tissue.

at some embodiments For example, the transformed Brassica plant is preferably non-sterile (i.e., fertile).

Preferably is the transformed Brassica plant, plant cell or Plant tissue able to seed with a transparent seed coat to deliver or is able to plants, the seeds with a transparent Seed Shell have to deliver.

The The present invention provides a method for increasing the Content of seed oil and protein and to reduce the levels of fibers in one Seed, the method comprising crossing a first Brassica plant, which has a CC genome coding for a first gene for a transparent gene Seed coat is homozygous, with the first gene for a transparent seed coat derived from a first AA genome of Brassica campestris, by using an embryo rescue technique, followed by cultivation in vitro, with a second Brassica plant carrying a second AA genome of Brassica campestris, which is for a second gene for a transparent seed coat is homozygous to a progeny Brassica plant to produce that for the first and second genes for transparent seed shells is heterozygous, and that with itself Cruising the offspring Brassica plant to produce the yellow seeded Brassica plant, the for the first and second genes for transparent seed coat is homozygous, and being the yellow-seeded Brassica plant produces seeds that are consistent and stable yellow color includes.

The method of the present invention may further comprise the steps of: (i) reading yellow seeds from black seeds or brown seeds and / or (ii) comparing the levels of Eru casein and glucosinolate (s) between the yellow seeds and the black seeds and the brown seeds.

A transformed Brassica napus plant that is capable of seed with a transparent seed coat is also here taught.

The The present invention discloses a seed oil or seed meal having an oil and protein content at least about 70% of the dried seed mass and with a Fiber content of not more than about 8% of the oil-free flour.

Preferably comprises the seed oil or the seed flour is an oil and protein content of about 70% to about 80% of the dried seed mass.

Preferably comprises the seed oil or the seed meal has a fiber content of not more than about 6% to about 8% of the oil-free Flour.

The The present invention discloses a use of an AA genome as a vector for the feeder of one or more genes of interest into a heterologous one Genome.

It Here, a transparent seed coat is taught by a gene for one transparent seed coat coded from NCIMB 40991 and / or NCIMB 40992 available is.

One or more among the transformed plant that transformed Cell or transformed tissue is prepared by applying the embryo rescue technique.

Preferably is one or more of the transformed plant that transformed Cell or the transformed tissue produced by a process, which comprises a step of chromosome doubling, wherein a means is used which causes the chromosome doubling, such as Colchicine.

Preferably is one or more of the transformed plant that transformed Cell or the transformed tissue produced by a process, which includes a stage of embryo rescue.

The present invention is advantageous since it is now possible To produce Brassica plants with yellow seeds. The present Invention is further advantageous as it is now possible Brassica napus plants to produce with yellow seeds.

Therefore we were in accordance with the present invention able to produce transformed plants with yellow seeds, such as B. transformed Brassica napus with yellow seeds.

In In the present application, the term "transformed" means cells and plants that are not naturally occurring, but instead through a human intervention selective crossing, preferably a method which is a biotechnological Includes stage (such as chromosome duplication and / or an embryo rescue technique) were manufactured.

The above aspects of the present invention will become something in more detail described.

in the Text means the term "yellow-seeded or with yellow seeds "one visible yellow embryo passing through a substantially transparent Seed Pod is produced.

in the Text means the term "transparent" that is a seed coat pigmentation essentially not compared to the seed coat of the wild-type is available.

in the Text means the term "stable" a yellow seed color, the consistent about transfer subsequent generations becomes.

Preferably the seed color becomes over transmitted consistently over at least two generations, preferably over at least three generations, preferably over at least four generations, preferably over at least five generations, preferably over at least six generations, more preferably at least seven generations.

In In the text, the term "consistent" essentially means one even distribution the yellow color.

Of the Term "lower Salary "in relation on the fatty acid erucic means less than 2% erucic acid.

Of the Term "middle Salary "in relation on the fatty acid erucic means more than 2% erucic acid and less than 40% erucic acid.

Of the Meaning "high content" in relation to the fatty acid erucic acid a content of more than 40% erucic acid.

Of the Term "lower Salary "in relation on glucosinolate (e) means a content of total glucosinolate (s) of less than 25 μmol / g Seeds.

Of the Term "middle Salary "in relation on glucosinolate (e) means a content of total glucosinolate (s) of more than 25μmol and less than 40μmol per g of seed.

Of the "High content" term in relation to glucosinolate (s) means a content of total glucosinolate (s) from bigger than 40μmol / g Seeds.

Upon achievement of the present invention, it was recognized that the major inhibitor of the attempt to grow B. napus plants with yellow seeds that could produce yellow seeds was the lack of a transparent seed coat gene. More specifically, it was recognized that a gene for a transparent seed coat was not present in the CC genome of B. napus. In a part of the analysis of the present invention which is by 1 it has been recognized that among the diploid Brassica species, forms with yellow seeds are found only in the species B. campestris (AA, 2n = 20), whereas among the amphidiploid species, variants with yellow seeds under B. carinata (BBCC , 2n = 34) and B. juncea (AABB, 2n = 36) can be found. The yellow seed in this species is a consequence of a transparent seed coat.

Indeed was in accordance surprisingly with the present invention found that it possible was a gene for a transparent seed coat in a plant, such. In their genome, to transfer, which did not have such a gene.

Of the Transfer can over Selective crossing procedures are carried out, which are certain essential or have preferred technical features. The details of this Techniques and procedures will be presented later.

To to the present invention were interspecific crosses at Brassica performed in different directions to the natural variants B. campestris, B. juncea and B. carinata in view of breeding of B. napus with yellow seeds to discover. So far, only limited Achievements achieved. For example, Barcikowska et al. (1987) and Zaman (1988) a species with CC genome and yellow seeds by developing the interspecific crossing of {yellow-seeded B. carinata x black-seeded B. alboglabra / B. oleracea} as one Intermediate in breeding of yellow-seeded B. napus were used in these experiments however, only partially obtained yellow seeds.

Liu (Liu 1983, Liu and Gao 1987) reported an attempt to breed one yellow-seeded B. napus, which was developed from the crossbreeding {B. napus x B. campestris (B. chinensis)}. However, the frequent occurrence indicated of black seeds and / or black spots on the shell as well the change the seed coat color due to environmental factors indicates that their yellow-seeded stems no over Generations were stable.

Zaman (1988) isolated a brown-seeded strain from an interspecific cross of {(B. carinata x B. campestris) x B. napus}. Chen (Chen et al., 1988; Chen and Heneen 1992) achieved yellow seeds in an F ₂ population derived from a cross between a resynthesized B. napus (resynthesized from B. campestris and a partially yellow seeded B. alboglabra) and a B. napus breeding line with yellow-brown seeds. However, the characteristic of the yellow seeds was not purebred, such as. B. to the F ₄ generation. Chef's partially yellow-seeded B. albogabra was developed from an interspecific cross between yellow-seeded B. carinata and black-seeded B. albogabra.

Rashid et al. (1994) reported their attempt to develop yellow-seeded plants from a slightly more complex interspecific Crossing of {[(B. napus x B. juncea) x B. napus] x [(B. napus x Carinata) x B. napus]}. However, it was about the stability of the seed coat color not reported in the resulting hybrids.

van Deynze (Van Deynze et al., 1993; Van Deynze and Pauls 1994) reported one partially yellow-seeded B. napus plant found at the University of Manitoba / Guelph was developed. However, it became an unstable yellow seed color and a significantly high seed coat pigmentation (black / dark brown Seeds) when the yellow-seeded strains are at a lower temperature from 16 ° C / 12 ° C (day / night) were drawn. This instability the yellow seed color allows such varieties for certain larger B. napus growing areas, such as Northern Europe and North America, appear unsuitable.

seaweed et al. (1997) developed yellow-seeded B. napus lines from various Breeding approaches, such as z. From interspecific crosses of {(B. campestris x B. oleracea) x B. napus}; {B. napus x B. juncea}; {B. napus x B. campestris} from irradiated progeny of B. napus and offspring of interspecific crossbreeds of B. napus. However, over the Stability of yellow seed color not reported.

It it is obvious that that Gene for a transparent seed coat of B. campestris, B. juncea and B. carinata in the prior art in various ways with regard to on breeding of yellow-seeded B. napus plants was used.

in the By contrast, our invention, inter alia, relates to transmission of the gene for the transparent seed coat over the AA genome of Yellow Sarson (B. campestris) in the CC genome of B. napus. Indirect evidence is provided that the transfer of the gene for the transparent seed coat is reached via allosyndese between the A and the C genome chromosomes. The resulting B. napus strain, the gene for the transparent seed coat of Yellow Sarson (B. campestris) in whose CC genome carries was used for the breeding of yellow-seeded oilseed rape.

To the For further background information, Attia (Attia and Röbbelen 1986; Attia et al. 1987) the meiotic configuration of chromosomes in: (i) amphihaploid AC, AB and BC, which were generated from crosses between three simple diploid species and (ii) in digenomic Triploids AAC, ACC, BBC and BCC, which were generated from crosses between the amphidiploid and the diploid species. In the amphihaploids 12.3 chiasms (7.3 II) were found. In AB, 5.8 chiasms (4.36 II) and in BC 2.0 chiasms (1.9 II) were found. In the digenomic triploids BBC and BCC, there was a dominance preferred pairing between the two homologous genomes, while the third single genome unpaired remained. On the other hand became a high frequency in the AAC and ACC triploids of allosyndetischer mating between the A and the C genome expressed by the formation of one or more trivalent in over 50% the pollen mother cells. According to Busso et al. (1987), became the homologous Mating between the A and C genome chromosomes in the trigenomic Haploid (ABC) is not disturbed by the presence of B genomic chromosomes.

These cytological observations clearly suggest that the A- and the C genome chromosomes closer to Brassica while related to each other the B genome chromosomes are phylogenetically removed from the A and C genome chromosomes. Accordingly, it was found in the amphihaploid genome constitution, that the gene transfer from the A to the C genome or vice versa.

One Process for the preparation of the transformed plants, namely by Use of the AA genome as a vector for the gene for the transparent seed coat is presented in the following examples.

at a highly preferred embodiment the present invention based on our knowledge that it is possible the gene for the transparent seed coat according to the present invention To use the invention to produce transformed cells.

Additionally disclosed the present invention also includes transformed plants containing the Gene for the transparent seed coat according to the present invention Invention.

The following examples were in accordance with the Budapest Treaty at the recognized depository The National Collections of Industrial and Marine Bacteria Limited (NCIMB) 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom AB24 3RY deposited on December 7, 1998.

Brassica napus 13-217 - deposit number NCIMB 40991

Brassica napus 13-219 - deposit number NCIMB 40992

The The present invention will now be described by way of example. wherein reference is made to the following figures:

1 , which is a schematic diagram,

2 , which is a schematic diagram,

3 , which is a schematic diagram,

4A , which is a schematic diagram,

4B , which is a schematic diagram,

5A , which is a schematic diagram,

5B , which is a schematic diagram,

6A which is a photographic representation

6B which is a photographic representation

7 , which represents a schematic diagram, and

8th which represents a photographic representation.

Something is more accurate 1 a schematic diagram that the occurrence of yellow seeds in the Brassica species.

2 Figure 13 is a schematic diagram showing the approach followed to transfer the gene for a transparent seed coat from the A to the C genome. The normal capital letters A, B and C indicate the three Brassica genomes. The superscript letter y indicates the gene for a transparent seed coat of Yellow Sarson (B. campestris). The superscript letter z indicates the gene for the transparent seed coat from B. carinata which, based on the gene for the transparent seed coat of Yellow Sarson (B. campestris), originate from different genomes. The superscript letter B indicates the gene for the black seed coat of the natural B. napus. The symbol * indicates the transfer via allosyndese of the transparent seed coat genes from the A to the C genome.

3 Figure 3 is a schematic diagram illustrating the process of development of yellow-seeded B. napus from interspecific crosses. The genome and seed coat labels are as for 2 described.

4A FIG. 3 is an HPLC chromatogram of an alkaline hydrolyzate of transparent seed shells (11A5156.082-K1217) of Brassica napus measured at 280 nm.

4B Fig. 11 is a HPLC chromatogram of an alkaline hydrolyzate of transparent seed coat (11A5156.082-K1217) from Brassica napus measured at 360nm.

5A Fig. 3 is a HPLC chromatogram of an alkaline hydrolyzate of black seed coat (Miro) of Brassica napus measured at 280nm.

5B Figure 3 is a HPLC chromatogram of an alkaline black seed coat (Miro) hydrolyzate of Brassica napus measured at 360nm.

6A is a photographic representation of seed shells of black seed of B. napus.

6B is a photographic representation of seed shells of yellow seeds of B. napus. The yellow seeds have a transparent seed coat.

7 Figure 3 is a graphical representation of a principal component analysis (PCA) of Agrovision color data from six seed samples (dark and yellow seeds) of Brassica napus.

8th demonstrates the color difference between yellow seeds (of the present invention) and black seeds (conventional type) of Brassica napus.

PROCEDURE AND PROCEDURES

The full Approach of the present invention is stepwise in the Stages I-VI indicated.

The stages I-II (which in 2 summarized) include the production of B. napus plants with yellowish-brown seeds bearing the genes for the transparent seed coat of Yellow Sarson (B. campestris) in their CC genome.

The stage III (the in 3 summarized) involves the resynthesis of the B. napus lineage carrying the genes for the transparent seed coat of Yellow Sarson (B. campestris) in its AA genome.

The level IV (which also in 3 summarized) includes the development of the yellow-seeded B. napus using the materials developed in stages I-II and III.

The Stage V includes the development of yellow-seeded B. napus of double low quality by crossing from yellow-seeded lines with conventional ones varieties of B. napus with double low quality.

The Stage VI includes the production of hexaploids, which is the claim of the present Invention supported.

Stage I: Interspecific crossing

Yellow-legged B. carinata x yellow-seeded yellow Sarson (B. campestris)

An interspecific cross between the yellow-seeded B. carinata (BBCC) line (accession number 381078) and the yellow-seeded Yellow Sarson (B. campestris) (AA) line originating in Bangladesh (accession number 3-0166.001) was performed and trigenomic haploid plants (ABC) were generated ( 2 ).

Stage II: Interspecific cross

Trigenomic F ₁ hybrid (ABC) x black-seeded B. napus (AACC)

The trigenomic haploids (ABC) of step I were crossed with black seeded natural B. napus (accession number 1-9007) and the resulting three way interspecific hybrids were propagated over a number of generations ( 2 ). These interspecific intersections were designed to assure allosynesis between the A and C genome chromosomes in the trigenomic haploids (ABC) and to ensure that subsequent increased generations would transfer the gene for the transparent seed coat from the A to the C genome.

The Steps I-II resulted in B. napus line # 06 with yellowish-brown Seeds.

Stage III: Interspecific crossing to Resynthesis of B. napus

Black-seeded B. alboglabra x yellow-seeded Yellow Sarson (B. campestris)

An interspecific cross between the black seeded B. alboglabra (CC) (Accession No. 381053) and the yellow seeded Yellow Sarson (B. campestris) (AA) (Accession No. 381049) was performed to resynthesize B. napus (AACC) ( 3 ). Since the reciprocal crossover was unable to produce any viable hybrid seeds in vitro, the use of an embryo rescue technique (described below) followed by an in vitro culture was used to obtain hybrid plants.

The Stage III resulted in the black seeded B. napus line # 1.

Stage IV: Generation of yellow seed B. napus

(B. napus with yellowish-brown seeds (No. 06) x black-seeded resynthesized B. napus (No. 01))

A conventional crossing was made between the lines No. 06 and No. 01 ( 3 ) and a pedigree breeding was added. In the F ₆ generation, completely yellowish-seeded lines were achieved.

The Stage IV resulted in the yellow-seeded B. napus lines 13-217 and 13-219.

Stage V: Development of yellow-seeded B. napus of twice lower quality

(zero erucic acid in seed oil and a little Glucosinolate (s) in the seed meal)

To develop a bivalve B. napus line of twice low quality (zero erucic acid and low glucosinolate (s)), yellow-seeded B. napus lines (13-217 and 13-219) were duplicated with conventional B. napus varieties low quality, such. B. Polo and Dakini crossed. A microspore culture method (Lichter 1989) was applied to all resulting F ₁ plants to generate double haploid (DH) lines. A yellow-seeded DH line with zero erucic acid was crossed with two (polys and Dakini) conventional B. napus varieties of twice the low quality to produce low-quality yellow-seeded B. napus. For this purpose, a pedigree breeding was then performed.

Stage VI: Generation of trigenomics hexaploid

(yellow-seeded B. carinata x yellow-seeded yellow Sarson (B. campestris))

Around the combined effect of genes for the transparent seed coat of B. carinata (BBCC) and Yellow Sarson B. campestris (AA) to determine seed coat pigmentation, were fertile trigenomic hexaploids (AABBCC) by chromosome doubling of trigenomic haploids (ABC) from yellow-seeded B. carinata x Yellow-seeded Yellow Sarson (B. campestris)} - plants produced from stage I.

Embryo rescue technique

• (i) die Schoten wurden zum richtigen Zeitpunkt der Entwicklung geerntet,
• (ii) die grünen Embryos (überlebende Embryos) wurden gerettet und wurden in vitro kultiviert, und
• (iii) die überlebenden Embryos wurden für die Sproßregeneration subkultiviert.

The stages for the embryo rescue technique were as follows:

• (i) the pods were harvested at the right time of development,
• (ii) the green embryos (surviving embryos) were rescued and cultured in vitro, and
• (iii) the surviving embryos were subcultured for shoot regeneration.

at a very specific embodiment have been B. cross-pollinated alboglabra plants with pollen from Yellow Sarson (B. campestris) and 24 to 28 days after pollination the immature pods were harvested, cut open and the hybrid embryos were rescued using the following sterile and cell culture procedures. The cut pods were surface sterilized with 70% Ethanol over 3 minutes followed by treatment with a 5% calcium hypochlorite solution over 20 minutes. The pods were washed three times with sterile water and the Length after under cut open in a stereo microscope. The developed (fertilized) Seed plants were excised and the reachable embryos were saved. These rescued embryos were incubated at 24 ° C in Murashige-Skoog medium (1962) 0.1mg NAA and 0.1mg kinetin per liter medium and under a continuous Lighting of 800 lux cultivated. After 20 to 30 days of culture became the survivors Embryos in the same medium for the shoot induction subcultured.

chromosome doubling

(a) Chromosome doubling of in in vitro - Culture regenerated sprouts

• (i) Die Sprosse wurden herausgeschnitten und wurden in einer wäßrigen Lösung von Colchicin untergetaucht,
• (ii) nach der Behandlung mit Colchicin wurden die Sprosse mit sterilem Wasser gewaschen und der untere Abschnitt des Stammes wurde durch Schneiden entfernt, und
• (iii) die Sprosse wurden in Wurzelmedium überführt.

The essential steps for the chromosome doubling technique were the following:

• (i) The shoots were excised and submerged in an aqueous solution of colchicine,
• (ii) after treatment with colchicine, the shoots were washed with sterile water and the lower portion of the strain was removed by cutting, and
• (iii) the shoots were transferred to rooting medium.

at a very specific embodiment have been Plants with duplicated chromosomes get through the treatment of regenerated sprouts in leaf stage 2-4 (unexpanded Leaf) with an aqueous solution of 0.5% colchicine plus 2.5% DMSO (modified version according to Gland (1981)). The shoots were in this solution submerged to the level of the meristem / shoot crest. After 16 to 20 hours, the shoots were taken out, with sterile Water washed three times and 3-5mm of the lower part of the shoot were cut off. The shoots were then in Murashige-Skoog medium (1962), supplemented with 3mg IBA per liter medium for transferred the root induction and under a lighting condition of 800 lux.

(b) Chromosome doubling of plant branches in vivo

• (i) in die Blattachseln wurde eine wäßrige Lösung von Colchicin injiziert,
• (ii) an der selben Stelle wurde in Abständen wiederholt eine Injektion verabreicht.

The essential steps for the chromosome doubling technique were the following:

• (i) an aqueous solution of colchicine was injected into the leaf axils,
• (ii) In the same place, an injection was repeatedly given at intervals.

at a very specific embodiment have been trigenomic hexaploid branches / shoots with doubled chromosomes obtained by adding trigenomic haploid (ABC) an aqueous solution of 0.5% colchicine plus 2.5% DMSO (modified version according to Gland (1981)) got injected. Slightly more accurately one injected in 38 Blattachseln of 19 plants three times the colchicine / DMSO solution at intervals of 24 hours. The hexaploid nature of the branches was confirmed by optical observation and analysis by flow cytometry the nuclear DNA. These branches produced fertile flowers, i. they exhibited viable pollen and produced viable seeds when propagated unlike the flowers from other branches (trigenomic haploid) that were sterile.

Analysis by flow cytometry

The Analysis by flow cytometry was performed by the procedures followed by Galbraith were followed et al. (1983) and Sabharwal and Dolezel (1993). For these purposes was nuclear DNA from seeds that have been raised from propagated seeds were used.

Isozyme electrophoresis

Leaves (about 3cm tall) of three Weekly plants were dissolved in 0.1M Tris-HCl buffer (pH 7.2) 0.5% DDT (1-4-dithio-DL-Threitol), homogenized and at 20,000 Rpm over Centrifuged for 1.5 minutes, after which the supernatant of the samples on a Filter paper with 9mm × 3mm (Whatman No. 1) was absorbed. A horizontal starch gel (13% hydrolyzed Strength, Sigma S-4501) was created and the samples were applied that she approximately Covered 1/3 of the gel width of the cathode end of the gel. The gel and the buffer systems in the shell were each 5 mM L-histidine monohydrochloride (adjusted to pH 7.0 with NaOH) and 0.4 M trisodium citrate dihydrate, 0.1 M citric acid, (pH 7.0). The electrophoresis was carried out for 3.5 hours at 200 V, 150 mA at 0 ° C, to ensure a suitable separation of the enzymes. All subsequent ones Färbeverfahren were according to Murphy et al. (1990).

Measurement of erucic acid

Of the Content of erucic acid in the seed oil was measured by gas chromatographic analysis using the method was followed by that of of the Association of Official Analytical Chemists (1990) has been. Typically, seed oil (triglycerides of fatty acids) was made extracted semisolid and methylated by adding methanol. The methyl esters of fatty acids were purified by gas chromatography using an apparatus of the type HP 5890 Series II analyzed, supported by a FID detector and an autosampler coupled to a ChemStation HP3365. The pillar was 50m long and 0.32mm wide and it was WCOT Quarzgut (Chrompack) with a stationary one Phase of CP-sil-58CB. Helium was used as a carrier gas with a flow rate from 276ml / min. There was an oven temperature of 205 ° C, an injection temperature of 250 ° C and a detection temperature of 270 ° C applied.

Measuring seminal glucosinolate (s) (GLS)

A quantitative measurement of total sucrose glucosinolate (s) (GLS) was performed using the method described by Smith et al. (1985). A qualitative measurement of the GLS contents in the seed and Two hundred microliters of distilled water were added to five crushed seeds and incubated at room temperature for 10 minutes using the glucose assay method. A medi-test glucose strip (manufactured by Macherey-Nagel) was introduced into the solution and the color change that occurred after 30 to 60 seconds was scored on a scale of 1 to 5.

Determination of the chemical Fingerprint of transparent seed shells compared to black Seed shells of B. napus

The seed shells were grown essentially in accordance with the method of Andreasen et al. 1998 (Journal of the Science of Food and Agriculture, in press) for the purpose of extraction of soluble and insoluble ester-bound phenolic acids analyzed. The seed coat of the line 11A5156.082-K1217 (transparent seed coat) ( 6B ) and the variety Miro (black seed coat) ( 6A ) was prepared by hand and further purified by a gravimetric method using a vertical airflow.

The seed shells (100 mg) were homogenized using a mortar, 100 ml of 1.0 M NaOH were added and incubated at room temperature for 18 hours under N ₂ in the dark. Then the pH of the hydrolysates was adjusted to below 2 with 1.0 M HCl, extracted three times with 40 ml of ethyl acetate. The combined ethyl acetate fractions were evaporated under vacuum at room temperature and the dried residue was dissolved in 3.0 mL of 50% acetonitrile and 2.0% trifluoroacetic acid and filtered through a 0.45 μm nylon filter.

The Extracts were analyzed by HPLC (Hitachi, Merck) which equipped were with a LiChroCART250-4 analytical RP-18 column (5μm), at 35 ° C below Use of a solvent gradient system consisting of solvent A (50% acetonitrile and 0.5% trifluoroacetic acid) and solvent B (8% acetonitrile and 0.5% trifluoroacetic acid) with the following elution profile: 0-10 min: 100% B, 11-20 min: 90% B, 21-30 min: 85% B, 31-45 min: 80%, 46-50 min: 75% B, 51-65 min: 100% A and 66-80 min: 100% B. Flow rate: 1 ml / min. Injection volume: 50 .mu.l. detection with a photodiode array detector at 280nm and 360nm.

image analysis

Six different semen samples were used for this purpose: sample number seed color 1 black 2 yellow 3 yellow 4 black 5 yellow 6 dark Brown

The Image analysis was performed with Agrovision Grain Check, where the reflection of red, green and blue light was measured as well as the total light reflection. The Data were analyzed using a multivariable analysis system, only the principal component analysis (PCA) was performed.

RESULTS

Stage I

Interspecific cross (B. carinata x Yellow Sarson (B. campestris)}

If the B. carinata line at the interspecific junction as the female plant was used, 3.28 hybrid seeds per pollination were obtained. The reciprocal cross gave 5.15 seeds per pollination. Although the degree of appropriate interspecific crossability between these two species was, was a larger number at hybrids (approx 66%) achieved by using the embryo rescue procedure. Therefore, the embryo rescue procedure is highly preferred for these purposes Feature of the present invention.

Morphology of trigenomic (ABC) F ₁ hybrid plants

The trigenomic F ₁ hybrids were easily distinguished from their parent lines by their morphological characteristics. For example, the leaf hairiness, stem encompassing of the stem and the yellow color of the petals of the F ₁ hybrids were comparable to the B. carinata line, whereas the leaf marginal perforation was comparable to that of Yellow Sarson (B. campestris). The confirmation of these interspecific hybrids was carried out using the isozyme electrophoresis method as already described.

Stage II

Crossability of trigenomic F ₁ hybrids (ABC) with B. napus (AACC)

The haploid genome composition of the trigenomic hybrids gave very little fertility. When cruising with conventional B. napus The hybrid plants produced about 0.025 hybrid seeds per pollination. About the same number of fertilized ovules were rejected before they reached full maturity.

Fertility of the interspecific Crossing ABC x AACC-derived hybrids

The pollen fertility of the hybrids produced from the crossing of trigenomic haploids (ABC) with natural B. napus (AACC) was only 16.6%. As a result, manual propagation of the individual plants, with pollen from fully developed flowers applied to the scar, was required to harvest a sufficient number of F ₂ seeds. All harvested seeds had a black seed coat.

Selection of seed coat color from ABC x AACC (F ₂ to F ₇ generations)

272 F ₂ plants were grown (Table 1). The pollen fertility of these plants varied from 0% to 79%. In order to avoid any cross-pollination, the flower cluster of all F ₂ plants was individually isolated with parchment paper bags. 106 (39%) of the F ₂ plants produced seeds when using this bag isolation procedure. Of these seed-bearing F ₂ plants, 97 had a black seed coat, while 9 had a brown seed coat. 116 F ₃ plants were grown on the 9 F ₂ families with brown seeds, of which 99 plants produced viable seeds under bag isolation. A single F ₃ family (# 0026,002) split between black, brown and slightly brown seeds, while the other eight families produced only black / brown seeds. The light brown-colored F ₄ seeds, which were produced on two F ₃ plants, were used for further selection, an improvement of the seed coat color with regard to obtaining a yellowish brown seed was only for the progeny of 1 (No. 0026,092) reached the 2 F ₃ plants. The progeny of the yellowish brown-colored seeds could not be completely stabilized in the following F ₅ and F ₆ generations. However, the F ₇ line (# 06) was crossed with yellowish-brown colored seeds with resynthesized B. napus line (# 01) (resynthesized from B. alboglabra x Yellow Sarson (B. campestris)) for development a purebred yellow-seeded B. napus plant.

Stage III: Interspecific crossing to Resynthesizing B. napus

(black-seeded B. alboglabra x yellow-seeded Yellow Sarson (B. campestris))

An interspecific cross between black-seeded B. alboglabra (CC) and yellow-seeded Yellow Sarson (B. campestris) (AA) was performed to resynthesize B. napus (AACC) ( 3 ). Since the reciprocal crosses were incapable of producing any viable hybrid seeds in vivo, an embryo rescue technique (described above) was used, followed by in vitro culture. Accordingly, the application of the embryo rescue method for the resynthesis of B. napus is considered to be an essential technical feature of the present invention. For these purposes, B. alboglabra was cross pollinated with Yellow Sarson (B. campestris) and about 0.45 embryos were saved per cross-pollinated pod, of which 40% survived under in vitro culture conditions and amphiphapoid (AC) plantlets led. The young plants were treated with an aqueous solution containing 0.2% colchicine plus 2.5% DMSO for the purpose of doubling the chromosomes. The resynthesized B. napus with doubled chromosomes was self-compatible in nature and had the trait "white petals" of the B. alboglabra lineage and produced black-shelled seeds when propagated.

Stage IV: Production of completely yellowish-seeded B. napus

(yellowish-brown-seedy B. napus (No. 06) x black-seeded resynthesized B. napus (# 01))

It became a conventional intersection between the lines No. 06 and No. 01 performed. The low content of seeds (about 10 seeds) per pollination (Table 2) was expected, as both parent lines exceeded those already described Interspecific intersections had been developed.

Of the increased seeds of 259 F ₂ plants examined visually for their color (Table 3), 199 (76.8%) produced black / brown colored seed hulls, 51 (19.7%) produced partially yellowed seeds and 9 (3.5%) produced yellow / brown colored seeds. Of the increased seeds of all 9 yellow-brown-seeded F ₂ plants, F ₃ families were produced. Of the 9 F ₃ families, 4 families were separated for black / brown seeds and partially yellow seeds in a 19:41 ratio, 4 families split into black / brown, partially yellow, and yellow-brown seeds in proportion from 19:48:62 and 1 F ₃ family split into partially yellow, yellow-brown and almost yellow seeds in a ratio of 9: 18: 1. Thus, 1 nearly yellow-seeded F ₃ plant was obtained from a total of 217 F ₃ plants from 9 families. The increased seeds of the individual nearly yellow-seeded and the selected 8 yellow-brown-seeded F ₃ plants were used to breed F ₄ families. In the F ₄ families, which were produced from the yellow-brownish-seeded F ₃ plants, no yellow-seeded plants were found. On the other hand, the F ₄ family, which had been produced from the almost yellow seeded F ₃ plant, again separated to partially yellow, yellow-brown and yellow seeds. The proportion of yellow-seeded plants (40.9%) in this generation was significantly higher than that obtained in the F ₃ generation. The F ₅ and F ₅ generation families were grown only from the selected yellow seeded plants. The separation into yellow-brown and yellow-colored seeds continued with dominance of the yellow-seeded plants in each subsequent generation. All the seeds that had been obtained in the F ₇ generation exhibited a consistent Us stable yellow color.

Because the yellow-seeded lines so developed were derived from a number of interspecific crosses, low fertility could be expected in these plants. Selection for improved fertility was made, as shown below, along with selection for a consistent and stable yellow seed color: the yellow seeded plants of the F ₄ to F ₉ generation were crossed with natural B. napus by the yellow-seeded plants were used as the female plants. The extent of crossability was in each successive generation from 3 seeds per pollination in the _F4 generation almost 7 seeds per pollination in the _F9 generation increased (Table 4).

The seed oil and the seed flour of the yellow-seeded B. napus, present in the present Each case contained an average salary on the fatty acid erucic (26-28%) and a high content of glucosinolate (s) (> 40μmol / g seed). Such two lines (13-217 and 13-219) were made with conventional oilseed rape B. napus of doubly low quality crossed to a yellow seeded oilseed rape As napus of doubly low quality to develop. The above two yellow-seeded B. napus lines had white petals, apparently derived from the C genome of B. alboglabra.

Stage V: Development of a yellow seeded oilseed rape B. napus of doubly low quality (zero erucic acid in the oil and little Glucosinolate (s) in the seed meal)

There were F ₁ hybrids of crosses between yellow-seeded lines (13-217 and 13-219) and conventional B. napus double low quality, such. For example, the polos and Dakini varieties are used to create double haploid (DH) plants / lines. 287 DH plants / lines were generated from four crosses (Table 5).

Inheritance of the seed coat color in the double haploid (DH) population

The following splitting patterns for seed color was found in 287 DH lines (Table 5): 195 DH lines had a dark black-brown seed color, 34 were reddish-brown, 27 were partly yellow, 14 were yellow-brown and 17 were yellowish-seeded. As the data from the DH lines with black / dark brown, reddish-brown, partially yellow and yellow-brown seeds were grouped into classes, a distribution pattern of 270: 17 was obtained, which is good in the splitting pattern of 15: 1 matched four gene loci. in the Contrary to this, when the data from the DH lines with the black / dark brown, reddish-brown and partially yellowed Seeds were grouped together, and the data from the DH lines with the yellow-brown and yellow-colored seeds into a second Class summarized were obtained, a splitting pattern of 256: 31, resulting in a splitting pattern from 7: 1 for 3 gene pairs matched. It would be only logical, the data from the latter two classes (yellow and yellow-brown classes) when the parental yellow-seeded Lines used at intersections under any circumstance would produce yellow-brown seeds. The propagation of 30 plants of these two parental yellow-seeded colored Lines grown in the field indicated seed coat color instability in 30% of the examined plants. However, such became one Instability not found when the plants were grown in a glasshouse. Even though the chi-square values that have been obtained are not valid for the four- still for that three-gene loci model were significant, was a better match with the splitting ratio (in terms of chi-square and p-values) for the four-gene loci model, which indicated that the transparent seed coat color for yellow seeds, the consequence of a homozygous recessive condition in is all four loci. As the seed coat color in B. campestris of two loci is determined and the transparent seed coat color (yellow seeds) is a consequence of the homozygous recessive condition in both loci (Stringham 1980, Schwetka 1982), suggest the results that it was on Most appropriate is a four-gene model for transparent seed coat color B. napus into consideration

Inheritance of petals and color erucic acid content in double haploid (DH) lines

A total of 61 double haploid lines from four crosses were tested for petal color and erucic acid content (Table 6). Twenty-six (26) lines had a yellow petal color and thirty-five (35) lines had a white petal color. This distribution ratio of 26:35 is consistent with a Mendelian splitting pattern of 1: 1 (X ² = 1.05, p = 0.5-0.3). The seeds of 27 DH lines were nearly free of erucic acid (range of 0.04-3.1%, mean 0.4%), while 34 lines had an erucic acid content in the range of 15.8-33.4% (mean of 22.7%). This distribution ratio of 27:34 is consistent with a Mendelian splitting of 1: 1 (X ² = 0.59, p = 0.5-0.3), suggesting that a single pair of functional alleles are responsible for the erucic acid content in the two yellow-seeded lines existed. All of the lines with white-blooded petals were associated with the presence of erucic acid, except for two lines (0.5 and 3.1% erucic acid) that were nearly free of erucic acid. Likewise, all yellow-blooded lines were associated with the absence of erucic acid except for a line containing 25.3% erucic acid. These results suggest that in the C genome, the gene locus controlling petal color and the locus controlling the erucic acid content are located on the same chromosome, but that recombination between these two loci can sometimes occur, as in the present case 4.9% of the DH lines occurred.

Inheritance of total glucosinolate (GLS) content in double haploid (DH) lines

Under Application of the method of Smith et al. (1985), were seeds of A total of 202 DH lines from the four intersections on their GLS content examined (Table 7). The seeds of 7 lines showed less than 20μmol GLS seeds per gram while the seeds of the remaining 195 lines more than 20μmol GLS per g seeds. This distribution of 7: 195 clearly agrees with the splitting pattern 1:15 for four gene loci. The splitting pattern in each individual crossing did not differ significantly from the overall pattern.

From the above-mentioned double haploid breeding program it was possible to obtain a B. napus line with yellow colored seeds (154.004) whose seeds are free of erucic acid and whose seed meal contained a high content of GLS. To lower the GLS content of the seeds from the yellow seeded B. napus, the DH line (154,044) was compared with conventional oilseed rape of twice low quality, such as. As Polo and Dakini, crossed. The F ₂ populations were grown in field plots. Seeds from a total of 144,914 open pollinated F ₂ plants were examined for their seed coat color, of which 640 plants were harvested from the openly pollinated seeds, as they were yellow-seeded or nearly yellow-seeded.

For the qualitative measurement of the GLS content in the seeds of these 640 yellow seeded plants, the glucose test method was used. Using the glucose test results, the seeds of 507 plants showing a very high test value (3-5) were evaluated as having a high GLS content and were discarded. A quantitative GLS measurement was performed with the seeds of the remaining 133 plants, which had a test value of 2. The seeds of 12 plants, which had a relatively low GLS content (25-40 μmol / g seeds) were used to produce F ₃ generation plants. A total of 600 F ₃ plants were grown, propagated and examined for their seed color. 287 plants produced yellow or yellow-brown or partially yellow-colored seeds, while the remaining 313 plants produced brown or black-stained seeds. Glucose assays were performed on the seeds from the 287 plants mentioned above, of which 87 yellow-24 yellow-brown and 23 partially yellow-seeded families were selected for the development of F ₄ generation plants. Of the 87 F ₄ families derived from the yellow seeded F ₃ plants, 40 families were stable with respect to the yellow seed color, while 47 F ₄ families split again with respect to yellow and yellowish-brown seed color. As expected, the splitting of the seed color was also observed in the progeny of 24 yellow-brown and 23 partially yellow-seeded F ₃ plants. The increased seeds of a total of 402 F ₄ plants were examined for seed colors and 114 were discarded due to their brown seeds. For the increased seeds of the remaining 288 F ₄ plants (yellow and yellow-brown and partly yellow seeds), quantitative measurements of GLS were performed. Seeds from 5 plants could be shown to have a GLS content of less than 20 μmol / g seed and to be yellow in color.

These Results indicate that yellow-seeded B. napus for the commercialization can be developed, these being different Levels of seed oil and seed meal quality may have, such. For example: (i) zero erucic acid and low glucosinolate (e), (ii) zero erucic acid and a lot of glucosinolate (s), (iii) a lot of erucic acid and a little glucosinolate (s), (iv) a lot of erucic acid and a lot of glucosinolate (s).

Step VI: Synthesis of trigenomic hexaploids (AABBCC) from the interspecific cross of B. carinata x Yellow Sarson (B. campestris) and their seed coat color

Trigenomic hexaploids were obtained by the injection of trigenomic haploids (ABC) with an aqueous solution of 2.5% DMSO plus 0.2% colchicine, which induces chromosome doubling ( 2 ). The 38 leaf axils from 19 plants injected with the colchicine / DMSO solution yielded 18% fertile, trigenomic hexaploid shoots (branches) that produced 5.4 to 35% viable pollen and were self-compatible. By manual propagation, a rate of approximately 93% pods was obtained and the number of viable seeds harvested per multiplication was 3.27. All propagated seeds were uniformly brown in color. The next generation of plants was grown from increased seed and the hexaploid nature of these plants was confirmed by flow cytometric analysis of nuclear DNA. The increased seeds produced by these plants were also brown.

Determination of the chemical fingerprint of transparent seed shells in comparison to black seed shells from B. napus

To determine whether the transparent appearance of the seed coat compared to black seed coat is the result of a reduction in the concentration of chromogenic substances, a chemical fingerprint was taken by HPLC of a basic seed coat hydrolyzate ( 4 - 5 ).

The chromatograms showed that some light-absorbing substances were present in much smaller amounts in the transparent seed shells compared to the black seed shells. For example, a substance measured at 280nm and having a retention time of 7.50-7.53 min eluted at a 35-fold lower concentration in the transparent seed coat than in the black seed coat ( 4A - 5A ). On average, the transparent seed shells contained 4.6-fold lower concentrations of substances that absorb at 280nm.

Comparable data were obtained by measuring the absorption at 360 nm, which was carried out simultaneously ( 4B - 5B ). For example, two compounds eluted at 17.04-17.11 and 26.78-26.82 min. Were found to be about 8-fold lower in the transparent seed coat compared to the black seed coat. Since these two compounds showed significant absorption at both 280nm and 360nm, they are likely to be flavonoids since this substance group having this characteristic absorption is often associated with the coloring of plant tissues, and they are preferably extracted by the applied analytical method (Norbaek et al., 1998, Phytochemistry, in press).

image analysis

In 7 a PCA plot of the Agrovision color data of the 6 seed samples is shown, with the first major component (x-axis) representing color intensity, which accounts for 91% of the total detected variation among the 6 seed samples. The present data delineate the yellow-seeded samples (Nos. 2, 3, and 5) from the remaining black (# 1 and 4) and brown (# 6) seed samples. Within the dark semen samples, slight variations were found, while there was no variation between the three yellow-seeded samples.

Oil-, Protein and fiber content in the yellow-seeded B. napus

Materials and methods

Two yellow-seeded F ₇ lines, 13-217 and 13-219, were planted with two black-seeded spring varieties B. napus cvs. "Polo" and "Dakini" crossed. Microspore culture technique (Lichter 1989) was used for the production of DH lines from F ₁ plants. Bulk seed samples of black, brown and yellow seed were prepared using a small amount of seeds from these three DH lines of the particular seed color type, and were used for the measurement of oil, protein and fiber content. The oil and protein content was measured by the near infrared reflection using a suitably calibrated InfraAlyzer 2000 (Bran + Luebbe) and the fiber content was measured by the method described by Stringam et al. (1974).

Results

The oil, protein and fiber data from bulk seed samples of black, brown and yellow seeded DH lines are presented in Table 8. The yellow-seeded sample had higher oil and protein contents than its black seeded counterpart. Table 8: Oil, protein and fiber content (in parentheses, relative values) of different colored mass seeding samples of double haploid lines of hybrid yellow-seeded x black-seeded B. napus. seed color Oil + protein% seeds dry matter Fiber% seeds Fiber% oil-free flour Black seeds 68.2 (100) 8.6 (100) 13.6 (100) Brown seeds 69.8 (102) 5.9 (69) 9.6 (71) Yellow seeds 70.4 (103) 3.9 (45) 6.1 (45)

The oil and protein content correlate negatively with each other (Grami et al., 1977, Bengtsson, 1985).

This indicates that the oil or Protein or both the oil as well as the protein content in yellow-seeded B. napus to a level can be raised, the higher is as with black-seeded varieties in that it continues to breed. The fiber content in the yellow seed as well as in the oil-free flour of yellow seeds was about 55% compared to black seeds or oil-free Reduced flour of black seeds.

DISCUSSION

Origin of the gene for the transparent Seed coat in the CC genome of yellow-seeded B. napus derived from the Junction No. 06 x No. 01 was derived

The Seed coat color of the parental B. napus line No. 01 (AACC), the black-seeded B. alboglabra (CC) x Yellow Sarson (B. campestris) (AA) is completely black. This line is wearing the gene for the transparent seed coat in their AA genome and they lack the gene for the transparent Seed coat in their CC genome. Therefore, the production is a completely yellowish B. napus from an intersection of No. 06 x No. 01 not possible without a gene for the transparent seed coat in the CC genome of line no. 06.

The Gene for the transparent seed coat in the CC genome of the B. napus line No. 06 is the gene for the transparent seed coat of Yellow Sarson (B. campestris), which by Allosyndese between the chromosomes of the A- and the C genome during the development of the line no. 06 through the interspecific intersection has been.

The possibility that this Gene for the transparent seed coat in the CC genome of No. 06 immediately derived from the B. carinata (BBCC) line was characterized excluded that was shown that the Synthesis of the trigenomic hexaploids (AABBCC) from the crosses yellow-seeded B. carinata x Yellow Sarson (B. campestris) not in the The situation was that a transparent seed coat color, i. yellow seeds, to create.

Furthermore in a separate study, the CC genomic yellow-seeded Species B. alboglabra resynthesized from the junction {(yellow-seeded B. carinata x black-seeded B. alboglabra) x black-seeded B. alboglabra). Using this partially yellow-seeded B. alboglabra (CC) and the yellow-seeded B. campestris Yellow Sarson (AA) was B. napus (AACC) resynthesized. The resynthesized B. napus was completely black seeded. A similar one Chen's approach (Chen et al 1988, Chen and Heneen 1992) fails to produce any yellow-seeded B. napus.

These results clearly show that: (i) the parents, the F ₇ generation, the yellowish-brown-seeded B. napus line (# 06) derived from the cross {{B. carinata x Yellow Sarson (B. campestris)} x B. napus} carries the transparent seed coat color of Yellow Sarson (B. campestris) in its CC genome and (ii) the transparent seed coat of B. carinata in Connection with the gene for the transparent seed coat of Yellow Sarson (B. campestris) is unable to produce yellow-seeded B. napus.

SUMMARY

The present invention, after all, provides transformed plants, which include a transparent seed coat, ready.

According to one preferred aspect, the present invention describes the development of a stable and consistent transparent seed peeling, i. yellow-seeded oilseed rape B. napus from interspecific crosses. In this regard was the gene for the transparent seed coat color of the AA genome of Yellow Sarson (B. campestris) in the CC genome of B. napus via Allosyndese between the Transferred chromosomes of the A and C genome. The resulting B. napus AACC genome containing the gene for the transparent seed coat from Yellow Sarson (B. campestris) yielded a consistent and stable yellow-seeded B. napus plant line.

As in particular can be seen from the teaching of the examples, is the application of the present invention not technically to a single plant variety limited.

Tabelle 2. Kreuzbarkeit zwischen der durch zwischenspezifische Kreuzung abgeleiteten resynthetisierten B. napus-Linien Nr. 06 und Nr. 01 und der natürlichen B. napus cv. Jaguar. Kreuzung Bestäubungszahl Samenzahl pro Bestäubung Nr. 06 x Nr. 01 Nr. 01 x Nr. 06 17 10 9.4 10.3 Nr. 06 x Jaguar Jaguar x Nr. 06 28 10 16.9 26.1 Nr. 01 x Jaguar Jaguar x Nr. 01 15 31 9.8 18.5

Tabelle 4. Kreuzbarkeit der gelbsamigen B. napus als weibliche Pflanze mit natürlicher B. napus Generation der gekreuzten gelbsamigen Familien Nummer der verwendeten gelbsamigen Familie Anzahl der Bestäubungen Samenzahl pro Bestäubung F4 1 30 3.0 F6 6 208 4.4 F7 2 55 4.8 F8 2 70 6.8 F9 3 55 6.9 Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope of the invention. While the invention has been described in terms of specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention should be apparent to those skilled in the art of plant biology and / or plant biotechnology and / or molecular plant biology and / or neighboring fields within the scope of the following claims. TABLES

Table 2. Crossability between resynthesized B. napus lines No. 06 and No. 01 derived from interspecific crossbreeding and the natural B. napus cv. Jaguar. crossing pollination number Number of seeds per pollination No. 06 x No. 01 No. 01 x No. 06 17 10 9.4 10.3 No. 06 x Jaguar Jaguar x No. 06 28 10 16.9 26.1 No. 01 x Jaguar Jaguar x No. 01 15 31 9.8 18.5

Table 4. Crossbreeding of yellow-seeded B. napus as a female plant with natural B. napus Generation of crossed yellow-seeded families Number of the yellow-seeded family used Number of pollinations Number of seeds per pollination F4 1 30 3.0 F6 6 208 4.4 F7 2 55 4.8 F8 2 70 6.8 F9 3 55 6.9

Table 5. Seed color split in double haploid (DH) lines from the cross between yellow seeded B. napus (13-217 and 13-219) and black seeded natural B. napus (Polo and Dakini). crossing Total DH lines Black / dark brown seeds Reddish-brown seeds partly yellow seeds Yellow-brown seeds Yellow seeds Splitting 15: 1 ^a Split 7: 1 ^b X ² p X ² p Polo x 13-717 75 52 8th 8th 3 4 0008 0.9-0.95 12:43 0.5-0.7 Dakini x 13-217 70 43 13 7 4 3 12:19 0.5-0.7 12:20 0.5-0.7 Polo x 13-219 75 52 5 7 4 7 0.75 0.3-0.5 12:15 0.5-0.7 Dakini x 13-219 67 48 8th 5 3 3 12:12 0.7-0.9 12:48 0.3-0.5 total 287 195 34 27 14 17 12:01 0.9-0.95 0.61 0.3-0.5 ^a splitting examined after summarizing data from black / dark brown, reddish-brown, partially yellow and yellow-brown seeds in one class and from yellow seeds to the other class ^b splitting examined after pooling data from black / dark brown, reddish-brown and partially yellow seeds in one class and yellow-brown and yellow seeds in the other class

Table 6. Split in Petal Color and Erucic Acid Content (C _{22: 1} ) in Double Haploid (DH) Lines of Jun. Namibian Junction Junctions (13-217 and 13-219) and Natural B. napus (Polo and Dakini). Yellow petals White petals - C _{22: 1} + C _{22: 1} - C _{22: 1} + 0221 Polo x 13-217 3 - - 7 Dakini x 13-217 10 - 1 16 Polo x 13-219 8th - 1 3 Dakini x 13-219 4 1 - 7 total 25 1 2 33

The Absence (3.1% or less) and the presence (15.8-33.4%) of Erucic acid content is by (-)- and (+) signs specified.

Table 7. Cleavage by glucosinolate (GLS) content (μmol / g seed) in double haploid lines of a cross between yellow seeded B. napus (13-217 and 13-219) and natural B. napus (Polo and Dakini). crossing <20 μmol GLS > 20 μmol GLS Splitting, 1:15 X ² p Polo x 13-217 3 51 0005 0.9-0.95 Dakini x 13-217 2 55 12:34 0.5-0.7 Polo x 13-219 1 44 0.65 0.3-0.5 Dakini x 13-219 1 45 0.70 0.3-0.5 total 7 195 2.22 0.1-0.2

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Claims (2)

A method of producing seeds having an increased content of seed oil and protein and a reduced content of fibers, the method comprising: a) crossing a B. carinata with yellow seeds and a B. campestris with yellow seeds to produce a three genome Haploid, crossing the three genome-containing haploid with B. napus and propagating the progeny with themselves to produce a Brassica progeny plant, characterized in that it further comprises: b) crossing a first Brassica plant producing a CC Genome, and a second Brassica plant having an AA genome of a Brassica campestris with yellow seeds, applying an embryo rescue technique, followed by culturing in vitro to produce a Brassica offspring and c) crossing the Brassica offspring plant from step (a ) with the Brassica progeny of step (b) and propagating the progeny with themselves to produce a Brassica plant with yellow seeds, where the Brassica plant produces seeds with yellow seeds that have a consistent and stable yellow color. A method of producing a Brassica plant, a plant cell or plant tissue by selective crossing, the method comprising: a) crossing a B. carinata with yellow seeds and a B. campestris with yellow seeds to a three genome haploid crossbred the three genome-containing haploid with B. napus and propagate the progeny with themselves to produce a Brassica offspring plant, characterized in that it further comprises: b) crossing a first Brassica plant having a CC genome and a second Brassica plant having an AA genome of a Brassica campestris with yellow seeds, followed by embryo rescue technique cultivating in vitro to produce a Brassica progeny; and c) crossing the Brassica progeny plant of step (a) with the Brassica progeny plant of step (b) and propagating the progeny with themselves to produce a Brassica plant with yellow seeds, the Brassica plant produces seeds with yellow seeds which have a consistent and stable yellow color.